Biomedical subjects

Florence M Hofman

Publications and source records attributed to Florence M Hofman.

15 recordsLinked to original sources

PubMed2006

Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its non-cyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC), in tumor cells in vitro and in vivo.

BACKGROUND: 2,5-Dimethyl-celecoxib (DMC) is a close structural analog of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib (Celebrex) that lacks COX-2-inhibitory function. However, despite its inability to block COX-2 activity, DMC is able to potently mimic the anti-tumor effects of celecoxib in vitro and in vivo, indicating that both of these drugs are able to involve targets other than COX-2 to exert their recognized cytotoxic effects. However, the molecular components that are involved in mediating these drugs' apoptosis-stimulatory consequences are incompletely understood. RESULTS: We present evidence that celecoxib and DMC are able to down-regulate the expression of survivin, an anti-apoptotic protein that is highly expressed in tumor cells and known to confer resistance of such cells to anti-cancer treatments. Suppression of survivin is specific to these two drugs, as other coxibs (valdecoxib, rofecoxib) or traditional NSAIDs (flurbiprofen, indomethacin, sulindac) do not affect survivin expression at similar concentrations. The extent of survivin down-regulation by celecoxib and DMC in different tumor cell lines is somewhat variable, but closely correlates with the degree of drug-induced growth inhibition and apoptosis. When combined with irinotecan, a widely used anticancer drug, celecoxib and DMC greatly enhance the cytotoxic effects of this drug, in keeping with a model that suppression of survivin may be beneficial to sensitize cancer cells to chemotherapy. Remarkably, these effects are not restricted to in vitro conditions, but also take place in tumors from drug-treated animals, where both drugs similarly repress survivin, induce apoptosis, and inhibit tumor growth in vivo. CONCLUSION: In consideration of survivin's recognized role as a custodian of tumor cell survival, our results suggest that celecoxib and DMC might exert their cytotoxic anti-tumor effects at least in part via the down-regulation of survivin - in a manner that does not require the inhibition of cyclooxygenase-2. Because inhibition of COX-2 appears to be negligible, it might be worthwhile to further evaluate DMC's potential as a non-coxib alternative to celecoxib for anti-cancer purposes.

Animals↗

PubMed2006

Enhancement of glioblastoma cell killing by combination treatment with temozolomide and tamoxifen or hypericin.

OBJECT: The chemotherapeutic agent temozolomide has demonstrated antitumor activity in patients with recurrent malignant glioma. Because responses are not enduring and recurrence is nearly universal, further improvements are urgently needed. METHODS: In an effort to increase the clinical activity of temozolomide, the authors investigated whether its antitumor activity could be enhanced by adding tamoxifen or hypericin, two drugs that are known to inhibit the activity of protein kinase C. Human glioblastoma multiforme cell lines A172 and LA567 were treated with combinations of temozolomide and tamoxifen or hypericin in vitro, and cell survival was analyzed using various methods. Tamoxifen and hypericin were able to greatly increase the growth-inhibitory and apoptosis-stimulatory potency of temozolomide via the downregulation of critical cell cycle-regulatory and prosurvival components. Furthermore, with the use of an in vivo xenograft mouse model, the authors demonstrated that hypericin was able to enhance the antiglioma effects of temozolomide in the in vivo setting as well. CONCLUSIONS: Taken together, analysis of the results indicated that combination therapy involving temozolomide and tamoxifen or hypericin potently inhibited tumor growth by inducing apoptosis and provided an effective means of treating malignant glioma.

Animals↗

PubMed2006

Characteristics of tumor-associated endothelial cells derived from glioblastoma multiforme.

Glioblastomas multiforme (GBMs) are highly vascular brain tumors characterized by abnormal vessel structures in vivo. This finding supports the theory that glioma-associated endothelial cells (ECs) have intrinsically different properties from ECs in normal human brain. Therefore, identification of the functional and phenotypic characteristics of tumor-associated ECs is essential for designing a rational antiangiogenic therapy. The GBM-associated ECs have a large, flat, and veil-like appearance, in contrast to normal ones, which are small and plump. Although the tumor ECs have the typical markers, they proliferate more slowly than these cell types in normal brain. The GBM-associated ECs are resistant to cytotoxic drugs, and they undergo less apoptosis than control cells. Also, GBM-associated ECs migrate faster than controls and constitutively produce high levels of growth factors such as endothelin-1, interleukin-8, and vascular endothelial growth factor. An understanding of these unique characteristics of glioma-associated ECs is important for the development of novel antiangiogenic agents that specifically target tumor-associated ECs in gliomas.

Angiogenesis Inhibitors↗

PubMed2006

Fibroblast growth factor-2-retargeted adenoviral vector for selective transduction of primary glioblastoma multiforme endothelial cells.

OBJECT: Adenovirus transduction in gene therapy is dependent on the expression of the coxsackie virus-adenovirus receptor (CAR) for initial binding and on the integrin receptors (avb3, avb5) for viral internalization. Low and variable expression of CAR may be responsible for the low transduction rates seen with native adenoviral vectors. The goal of this study was to demonstrate increased transduction efficiency by retargeting the adenovirus with a fibroblast growth factor (FGF) ligand, FGF-2. METHODS: The retargeted adenoviruses were used to transduce human glioblastoma multiforme (GBM)-derived ECs (tumor-associated brain endothelial cells [TuBECs]), in which there is minimal CAR expression but a high expression of FGF receptor (FGFR). The results demonstrate that the transduction efficiency of TuBECs can reach as high as 80% when one uses an FGF2-conjugated adenovirus containing green fluorescent protein (FGF2-AdGFP) yet be only 5% when one uses the native adenovirus (AdGFP). The TuBECs were transduced with either a native adenovirus (AdHSV-TK) or a retargeted adenovirus (FGF2-AdHSV-TK), both of which carry the suicide herpes simplex virus-thymidine kinase (HSV-TK) gene. Administered as a cytotoxic prodrug, ganciclovir induced a significant decline in the proliferation rate and increased apoptosis in TuBECs treated with the retargeted adenovirus, compared with its effect on TuBECs treated with the native adenovirus. Increased transduction efficiency was determined by performing GFP-based flow cytometry, and the expression of the TK protein by the retargeted adenovirus was assessed by performing an immunohistochemical analysis focused on HSV-TK. The mechanism of cytotoxicity was determined to be apoptosis by performing a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. CONCLUSIONS: Fibroblast growth factor-2-retargeted adenoviral vectors may be used to increase the transduction of GBM-derived endothelial cells, enabling a new and efficient antiangiogenesis strategy for the treatment of malignant gliomas.

Adenoviridae↗

PubMed2006

Multiple signaling pathways are involved in endothelin-1-induced brain endothelial cell migration.

We have observed that the vasoactive peptide endothelin-1 is a potent inducer of migration of primary human brain-derived microvascular endothelial cells. By blocking signal transduction pathways with specific inhibitors, and using dominant negative mutant infections, we have demonstrated that multiple pathways are involved in endothelin-1-induced migration. Absolutely required for migration are protein tyrosine kinase Src, Ras, protein kinase C (PKC), phosphatidylinositol 3-kinase, ERK, and JNK; partial requirements were exhibited by cAMP-activated protein kinase and p38 kinase. Partial elucidation of the signal transduction sequences showed that the MAPKs ERK, JNK, and p38 are positioned downstream of both PKC and cAMP-activated protein kinase in the signal transduction scheme. The results show that human brain endothelial cell migration has distinct characteristics, different from cells derived from other vascular beds, or from other species, often used as model systems. Furthermore, the results indicate that endothelin-1, secreted by many tumors, is an important contributor to tumor-produced proangiogenic microenvironment. This growth factor has been associated with increased microvessel density in tumors and is responsible for endothelial cell proliferation, migration, invasion, and tubule formation. Because many signal transduction pathways investigated in this study are potential or current targets for anti-angiogenesis therapy, these results are of critical importance for designing physiological antiangiogenic protocols.

Brain↗

PubMed2005

Interleukin-8 differentially regulates migration of tumor-associated and normal human brain endothelial cells.

Interleukin-8 (IL-8) is a chemokine involved in angiogenesis, a process vital to tumor growth. Previously, we showed that endothelial cells derived from human tumor tissue have different functional and phenotypic properties compared with normal endothelial cells. This study analyzes the role of IL-8 in regulating angiogenesis of tumor-associated brain endothelial cells (TuBEC). Results show that TuBECs have a higher baseline migration rate compared with normal brain endothelial cells (BEC). TuBECs are unaffected when stimulated with IL-8 whereas BECs are activated. This lack of response of TuBECs to IL-8 is due to the constitutive production of IL-8. Endogenously produced IL-8 activates TuBECs in an autocrine manner as shown by IL-8 receptor inhibition. Blocking either CXCR1 or CXCR2 partially reduces TuBEC migration, whereas blocking both receptors further reduces migration. Treatment with antibody against vascular endothelial growth factor (VEGF) shows that production of IL-8 by TuBECs is dependent on VEGF. Transforming growth factor-beta1 (TGF-beta1), shown to down-regulate IL-8 production in BECs, does not inhibit IL-8 production in TuBECs. In summary, these studies show that TuBECs constitutively secrete IL-8 and autocrine activation by IL-8 is the result of VEGF stimulation. Furthermore, TuBECs do not respond to the feedback inhibition normally induced by TGF-beta1. These data emphasize the functional uniqueness of TuBECs. Understanding the functions and regulatory processes of tumor-associated endothelial cells is critical for developing appropriate antiangiogenic therapies.

Brain↗

PubMed2005

Multitarget inhibition of drug-resistant multiple myeloma cell lines by dimethyl-celecoxib (DMC), a non-COX-2 inhibitory analog of celecoxib.

2,5-Dimethyl-celecoxib (DMC) is a close structural analog of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib that lacks COX-2 inhibitory function. We and others have demonstrated that DMC, despite its inability to block COX-2, is able to potently mimic the antitumor effects of celecoxib in vitro and in vivo. In this current study, we investigated whether DMC would also be able to inhibit the growth of highly drug-resistant tumor cell variants. We focused on human multiple myeloma (MM) cells, as patients with MM frequently develop drug-resistant disease and ultimately succumb to death. Here we show that DMC (and celecoxib) inhibits the proliferation of various multiple myeloma cell lines, including several (multi) drug-resistant variants. Growth inhibition in drug-sensitive and drug-resistant cells is mediated via multiple effects, which include diminished signal transducer and activator of transcription 3 (STAT-3) and mitogen-activated protein (MAP) kinase kinase (MEK) activity, reduced expression of survivin and various cyclins, and is followed by apoptotic cell death. Thus, our study demonstrates that inhibition of proliferation and induction of apoptosis by DMC (and celecoxib) can be accomplished even in highly drug-resistant multiple myeloma cells, and that this effect is achieved via the blockage of multiple targets that are critical for multiple myeloma cell growth and survival.

Cell Line, Tumor↗

PubMed2005

Dimethyl-celecoxib (DMC), a derivative of celecoxib that lacks cyclooxygenase-2-inhibitory function, potently mimics the anti-tumor effects of celecoxib on Burkitt's lymphoma in vitro and in vivo.

The nonsteroidal anti-inflammatory drug (NSAID) celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor that has shown some promising results as an anti-cancer drug. However, the question arose as to whether or not its COX-2-inhibitory function is required for its anti-tumorigenic properties. We therefore employed dimethyl-celecoxib (DMC), which is a close structural analog of celecoxib that lacks COX-2-inhibitory function, to investigate this question. By performing a combination of in vitro and in vivo studies with Burkitt's lymphoma cells, we found that DMC potently mimics all of the anti-proliferative and anti-tumorigenic effects of celecoxib. In cell culture, DMC effectively inhibits cell proliferation through the down-regulation of cyclins A and B and the ensuing loss of cyclin-dependent kinase activity. This effect appears to take place in vivo as well and results in significantly (p<.002) reduced tumor growth in experimental animals. Thus, our results demonstrate that the anti-proliferative and anti-tumorigenic properties of celecoxib and DMC are indistinguishable, at least in Burkitt's lymphoma cells, and therefore, that the COX-2-inhibitory function is not required for these effects.

Animals↗

PubMed2005

CD8+-dependent CNS demyelination following ocular infection of mice with a recombinant HSV-1 expressing murine IL-2.

Demyelinating diseases comprise a spectrum of immunopathologic syndromes in which myelin, the fatty covering of nerve cell fibers in the brain and spinal cord, is destroyed. In this study, we have shown for the first time that ocular infection of BALB/c mice with a recombinant herpes simplex virus type 1 (HSV-1) expressing IL-2 (HSV-IL-2) results in CNS demyelination as determined by light microscopy and EM. The demyelinated lesions involve periventricular white matter, brain stem, and spinal cord white matter. Demyelination was detected in the CNS of infected mice up to 75 days (the longest time point tested) post HSV-IL-2 infection. In contrast, mice infected with HSV-IFN-gamma or HSV-IL-4, which are identical to HSV-IL-2 but express IFN-gamma or IL-4 instead of IL-2, did not exhibit demyelination. Control mice infected with wild-type HSV-1 or parental virus also remained free of these symptoms. During early times (days 3-7), post-infection with HSV-IL-2 virus, a T(H)1 + T(H)2 pattern of cytokines was produced by lymphocytes of infected mice while mice infected with HSV-IFN-gamma or control viruses produced a T(H)1 pattern of cytokine. By day 21 post-infection, all infected groups exhibited a T(H)1 pattern of response. Immunohistochemistry and FACS analyses of infiltrates in the brains and spinal cords of HSV-IL-2-infected mice showed elevations in CD4+ and CD8+ T cells and macrophages. However, T cell depletion studies suggest that only central memory CD8+ T cells are directly involved in the demyelination process, with macrophages being involved through a bystander effect.

Animals↗

PubMed2005

Functional and phenotypic differences between glioblastoma multiforme-derived and normal human brain endothelial cells.

OBJECT: Glioblastomas multiforme (GBMs) are hypervascular tumors characterized by endothelial cell (EC) proliferation. There is increasing evidence that ECs that infiltrate systemic tumors are different from normal blood vessel cells; whether this difference is seen in the central nervous system between GBM and normal brain tissue is not known. The goal of this investigation was to characterize and compare the functional and phenotypic properties of GBM-associated ECs and normal brain ECs. METHODS: Human ECs were isolated from fresh tissue specimens, purified using flow cytometry, and characterized by immunostaining. Proliferation was measured by determining bromodeoxyuridine incorporation and Ki-67 staining, and by performing the monotetrazolium assay. The migration rate of the cells was determined using the modified Boyden chamber technique. Apoptosis was evaluated by performing the TUNEL assay, cell death enzyme-linked immunosorbent assay (ELISA), and annexin V staining. Growth factor production was analyzed using the ELISA technique. The brain tumor ECs differed from normal brain ECs morphologically and by their expression and distribution of specific markers (that is, vascular endothelial cadherin [VE-cadherin] and CD31). Functional differences between the two cell populations were also evident. The brain tumor ECs proliferated more slowly and underwent less apoptosis than normal brain ECs; however, the tumor ECs migrated faster than the normal ECs. The normal ECs were sensitive to growth factors such as vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1), whereas the tumor ECs were not. In addition, the brain tumor ECs constitutively produced higher levels of ET-1 and VEGF, compared with the normal ECs. CONCLUSIONS: The data demonstrated that ECs derived from normal brain and from GBMs have significant phenotypic and functional distinctions. Further characterization of brain tumor ECs is essential for efficient antiangiogenic treatment of gliomas.

Apoptosis↗

PubMed2004

Extra-cellular superoxide promotes T cell expansion through inactivation of nitric oxide.

The mechanism and regulation of immunosuppression by nitric oxide (NO) is unclear. Extra-cellular superoxide (EC-O2-) production by NADPH-oxidase (phox) may prevent NO-mediated suppression of T cell proliferation. p47(phox-/-) mice are resistant to experimental allergic encephalomyelitis (EAE), coinciding with enhanced splenic NO activity, but no causal link was established. Here, we demonstrate such link, since p47(phox-/-) mice developed severe EAE by adoptive transfer, but only if NO production during ex vivo donor cell reactivation was inhibited. EC-O2- production increased during cognate T cell reactivation, while inhibition of EC-O2- by exogenous superoxide dismutase enhanced NO activity. By inhibiting NO, EC-O2- production promotes T cell expansion during peripheral immune-response activation, not during tissue inflammation.

Adoptive Transfer↗

PubMed2003

A simple method for isolation and characterization of mouse brain microvascular endothelial cells.

Brain endothelial cells, a site of the blood-brain barrier in vivo, regulate a number of physiological and pathophysiological processes in the brain including transport of nutrients, export of critical toxins, transmigration of circulating leukocytes and formation of new blood vessels. In this report, we describe a simple and reproducible method to isolate pure (>99%), functionally active endothelial cells from small quantities of adult mouse brain tissue. In vitro, these cells express typical phenotypic markers of differentiated brain endothelium such as von Willebrand factor, multiple drug resistant protein and glucose transporter-1, demonstrate uptake of acetylated low-density lipoprotein, and possess morphological and ultrastructural characteristics of microvascular endothelium. They form tight junctions and capillary-like tubes when stimulated by growth factors in an in vitro angiogenesis assay. In response to tumor necrosis factor-alpha, isolated mouse brain endothelial cells (MBEC) express vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). The protocol described here provides an effective and reliable method to isolate pure cerebral endothelium from adult mouse brain that should offer a useful tool for studying the role of altered vascular biology in mice with genetically manipulated brain disorders.

Animals↗

PubMed2003

Tissue expression of inducible nitric oxide synthase requires IFN-gamma production by infiltrating splenic T cells: more evidence for immunosuppression by nitric oxide.

The cause of increased severity of experimental allergic encephalomyelitis (EAE) in inducible nitric oxide synthase (iNOS)(-/-) or IFN-gamma(-/-) mice remains unclear. Transient chimeras were generated to examine the source of iNOS-inducing IFN-gamma in the central nervous system (CNS). IFNgamma(-/-) donor cells induced severe EAE but no iNOS expression in the CNS of wild-type recipients upon their immunization. By contrast, milder EAE, but strong iNOS expression was induced in immunized recipients of wild-type donor spleen cells. These results demonstrate that IFN-gamma secretion by infiltrating spleen cells is essential for iNOS expression during CNS inflammation. Furthermore, splenic NO is immunosuppressive.

Animals↗

PubMedDate not supplied

Involvement of CD8+ T-cells in exacerbation of corneal scarring in mice.

PURPOSE: To determine the specific immune responses involved in the exacerbation of corneal scarring induced by HSV-1 in gK vaccinated mice. MATERIALS AND METHODS: BALB/c mice were vaccinated with HSV-1 glycoprotein K (gK) and ocularly challenged with HSV-1. Infiltration into the cornea of T cells and macrophages was monitored by immunocytochemistry, and the effect of depletion of CD4+ T-cells, CD8+ T-cells, or macrophages on corneal scarring was determined. RESULTS: Following ocular challenge, CD4+ and CD8+ T-cells and macrophages were more abundant in the corneas of gK-vaccinated mice than in the corneas of mock vaccinated mice. Depletion of CD8+ T-cells, but not of CD4+ T-cells or macrophages, reduced the severity of corneal scarring in gK-vaccinated mice. CONCLUSIONS: We have shown that gK vaccination causes an overall increase in T cells and macrophages in the cornea after ocular HSV-1 challenge. The immunopathology induced by gK vaccination appears to be related to CD8+ T-cell activity, as depletion of these cells, but not other immune cells, reduced corneal scarring.

Animals↗

PubMedDate not supplied

The type IV phosphodiesterase inhibitor rolipram induces expression of the cell cycle inhibitors p21(Cip1) and p27(Kip1), resulting in growth inhibition, increased differentiation, and subsequent apoptosis of malignant A-172 glioma cells.

Upregulation of the cAMP/protein kinase A (PKA) pathway has been shown to result in decreased proliferation, increased differentiation, and subsequent apoptosis of malignant glioma cells. Conventional cAMP analogs, however, are difficult to use in a clinical setting. Therefore, we investigated the effects of rolipram, a drug that has undergone clinical trials as an antidepressant and has also been proposed as a treatment for multiple sclerosis. Rolipram acts as a specific inhibitor of type IV phosphodiesterase (PDE4), leading to increased intracellular levels of cAMP. We report that the inhibition of PDE4 by rolipram results in the activation of the cAMP/PKA pathway, with potent stimulation of a reporter gene containing a cAMP-responsive element in its promoter region. Further, treatment of the human glioma cell line A-172 with rolipram results in increased expression of the cell cycle inhibitors p21(Cip1) and p27(KiP1), and decreased activity of cdk2, a cyclin-dependent kinase essential for cell cycle progression. As a result, the proliferation of A-172 cells is inhibited, with induction of a Gl block. Eventually, rolipram-treated A-172 cells undergo differentiation, which is followed by apoptotic cell death. As we observe this effect with other glioma cell cultures as well, our results suggest that rolipram could prove useful as a novel differentiating agent with both cytostatic and cytotoxic potential in the treatment of malignant gliomas.

3',5'-Cyclic-AMP Phosphodiesterases↗